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. Author manuscript; available in PMC: 2015 Jan 8.

Published in final edited form as: Pathog Dis. 2013 Nov 5;70(2):185–188. doi: 10.1111/2049-632X.12102

Identification and characterization of MuiA. A) MTP87 cosmid was subjected to in vitro transposon mutagenesis to generate random gene knockouts. Shown in the inset are PAO579 (muc-23) exconjugants carrying cosmid MTP87 randomly mutagenized with an EZ::TN transposon (Epicentre), selected on a PIA plate supplemented with the appropriate antibiotic, and incubated at 37°C for 48h. B) Restriction map, gene organization and Tn insertion in the muiA gene. Homology of MuiA with its orthologs. Shown are the most homologous regions (1, 2 and 3): R. capsulatus (RC; ORF1654; 534 aa), B. japonicum (BJ; CAC38742; 560 aa), Nostoc sp. (NOS; NP_484904; 545 aa), and C. elegans (CE; NP_500427; 556 aa). A single Tn insertion occurred before regions 1, 2 and 3. C) PAO1 pHERD20T, PAO579 pHERD20T (vector control), PAO579 pHERD20T-muiA (wild-type muiA) and PAO579 pHERD20T-muiAΔN22 (deletion of N terminal signal sequence) were grown on PIA plates supplemented with carbenicillin and 0.1% arabinose for 24h at 37°C then for 24h at room temperature. The alginate was collected and measured using the standard carbazole assay. The values are representative of three independent experiments. Statistical significance was determine using the Student’s t-test in comparison with PAO579 (*P<0.05). D) The β-galactosidase activity of the algD promoter was measured using PAO1 and PAO579 miniCTX-P_algD-lacZ with pHERD20T, pHERD20T-muiA or pHERD20T-muiAΔN22. All strains were incubated at 37° C on PIA plates supplemented with tetracycline, carbenicillin and 0.1% arabinose. The values for the mean and standard deviation Miller Units (One Miller Unit=1000 X (A₄₂₀/−1.75 X A₅₅₀/OD₆₀₀ mL⁻¹ min⁻¹)) are shown as relative expression as compared to PAO1, and are representative of three independent experiments. Statistical significance was determine using the Student’s t-test in comparison with PAO579 (*P<0.05). E) Alginate measurements for various laboratory and clinical strains expressing pHERD20T (vector control) or pHERD20T-muiA in trans. Strains were cultured on PIA plates supplemented with 300 μg/mL of carbenicillin and 0.1% arabinose and incubated for 24h at 37°C then for 24h at room temperature. The values are representative of three independent experiments. Statistical significance was determine using the Student’s t-test in comparison with the vector control (*P<0.05).

Identification and characterization of MuiA. A) MTP87 cosmid was subjected to in vitro transposon mutagenesis to generate random gene knockouts. Shown in the inset are PAO579 (muc-23) exconjugants carrying cosmid MTP87 randomly mutagenized with an EZ::TN transposon (Epicentre), selected on a PIA plate supplemented with the appropriate antibiotic, and incubated at 37°C for 48h. B) Restriction map, gene organization and Tn insertion in the muiA gene. Homology of MuiA with its orthologs. Shown are the most homologous regions (1, 2 and 3): R. capsulatus (RC; ORF1654; 534 aa), B. japonicum (BJ; CAC38742; 560 aa), Nostoc sp. (NOS; NP_484904; 545 aa), and C. elegans (CE; NP_500427; 556 aa). A single Tn insertion occurred before regions 1, 2 and 3. C) PAO1 pHERD20T, PAO579 pHERD20T (vector control), PAO579 pHERD20T-muiA (wild-type muiA) and PAO579 pHERD20T-muiAΔN22 (deletion of N terminal signal sequence) were grown on PIA plates supplemented with carbenicillin and 0.1% arabinose for 24h at 37°C then for 24h at room temperature. The alginate was collected and measured using the standard carbazole assay. The values are representative of three independent experiments. Statistical significance was determine using the Student’s t-test in comparison with PAO579 (*P<0.05). D) The β-galactosidase activity of the algD promoter was measured using PAO1 and PAO579 miniCTX-P_algD-lacZ with pHERD20T, pHERD20T-muiA or pHERD20T-muiAΔN22. All strains were incubated at 37° C on PIA plates supplemented with tetracycline, carbenicillin and 0.1% arabinose. The values for the mean and standard deviation Miller Units (One Miller Unit=1000 X (A₄₂₀/−1.75 X A₅₅₀/OD₆₀₀ mL⁻¹ min⁻¹)) are shown as relative expression as compared to PAO1, and are representative of three independent experiments. Statistical significance was determine using the Student’s t-test in comparison with PAO579 (*P<0.05). E) Alginate measurements for various laboratory and clinical strains expressing pHERD20T (vector control) or pHERD20T-muiA in trans. Strains were cultured on PIA plates supplemented with 300 μg/mL of carbenicillin and 0.1% arabinose and incubated for 24h at 37°C then for 24h at room temperature. The values are representative of three independent experiments. Statistical significance was determine using the Student’s t-test in comparison with the vector control (*P<0.05).